Studies on Charcoal Rot of Infected by Macrophomina Phaseolina from Various Regions in Madhya Pradesh

Since there are various biotic and abiotic factor that is responsible for causing deficiency and diseases. It leads to the reduction of yield and health decreases in gross global productivity. Fungal pathogens like Macrophomina phaseolina is soil, seed and stubble borne pathogenic fungus (Khan, 2007). Now a day it has emerged as severely damaging fungus to the agricultural world. It belongs to the family Botryosphaericeae known as anamorphic fungus in the ascomycete group (Creus et al., 2006). Its sustainability in broad tropical to subtropical regions from and to semi-arid climates favors the inhabitation of fungus in various agro-climatic conditions including Asiatic nations (Diourte et al., 1995; Wrather et al., 2001). In India there are 15 meso agro climatic and 73 subzone. The evidences explain it sustains in soil in latent-state. Initiation of the microorganism as fungal pathogen accelerates in when plant is in optimal stress condition, Macrophomina phaseolina produces asexual structures have microsclerotia and pycnidia, which can be detected in soil and host tissue utilizing various techniques and assays (Babu et al., 2011), Microsclerotia can survive in soil for 2-15 yrs. or in root debris for longer periods (Baird et al., 2003; Sarr et al., 2014). Soybean (Glycine max (L.) Merrill) is one of the most economically and nutritionally significant leguminous oilseed crops cultivated worldwide. Believed to have originated in China, it was considered one of the five sacred grains vital to the foundation of early Chinese civilization (Hymowitz, 1970). In India, soybean gained substantial commercial importance over the past six decades, especially in central and western regions. Soybean seeds are rich in macronutrients, containing approximately 40% high-quality protein and 20% edible oil (Talukdar et al., 2009). In addition to these, soybean is a dense source of essential amino acids, minerals like calcium and phosphorus, and fat-soluble vitamins including A, B-complex, C, and D. The crop also contains antioxidants and nutraceutical compounds-particularly is flavones-that have been shown to reduce blood cholesterol levels, enhance immune function, and lower the risk of cardiovascular disease, diabetes, and hormone-related cancers (Sharma et al., 2008; Kumar et al., 2014; Liu, 2010). These qualities have established soybean as a vital component of functional foods and health-promoting diets globally.

Life Cycle of Macrophomina phaseolina

Macrophomina phaseolina is a fungal pathogen that causes charcoal rot disease in various plants, including soybeans, maize, sunflowers, and other economically important crops. Understanding its life cycle is crucial for devising effective management strategies (Agarwal et al., 2013; Islam et al., 2018).

5. Survival as Sclerotia: The primary survival and overwintering structure of Macrophomina phaseolina is sclerotia. These are compact masses of fungal mycelium and are capable of surviving adverse conditions in the soil for extended periods (Kaur et al., 2012).
5. Germination: Under the favorable conditions like adequate moisture and temperature, sclerotia germinate, forming mycelium.
5. Infection: The fungus can infect host plants directly through roots or lower stems. Infection is facilitated by fungal penetration of plant tissues.
5. Colonization and Spread: Macrophomina phaseolina colonizes the vascular tissues of the host plant, causing the characteristic symptoms of charcoal rot disease, including wilting, stem necrosis, and vascular discoloration.
5. Production of Sclerotia: As the disease progresses, the fungus produces new sclerotia within the infected plant tissues.
5. Release of Sclerotia: When infected plant tissues senesce and decompose, sclerotia are released into the soil.
5. Repeat Cycle: The released sclerotia serve as a source of primary inoculum for subsequent infection cycles, completing the life cycle.

The fungal isolates were further purified and identified based on morphological characteristics and pathogenicity tests. Morphological examination revealed the diversity of pathogens infecting soybean, highlighting variations in structure and appearance among different pathogen types. Morphological studies contribute to understanding the interactions between pathogens and their host plants. Certain morphological traits may correlate with pathogenicity virulence and host specificity.        

MATERIALS AND METHODS

The present investigation was conducted on studies on charcoal rot of Soybean caused by Macrophomina phaseolina (Tassi) Goid and its Management included survey for charcoal rot, isolation, purification, pathogenicity, morphological variability and physiological studies, pathogen. The laboratory experiments were conducted in Department of Biological Sciences, Rani Durgavati University, Jabalpur (M.P.) and pot experiment was undertaken in green house.

Survey and Collection of Infected Soybean Crops from different regions in Madhya Pradesh

Study Area

Survey was undertaken in the major field soybean crops region of Madhya-Pradesh during 2016-17 and 2017-18 based on the information provided by the extension workers and reports of Indian Council Agriculture Research (ICAR, M.P.) and Jawaharlal Nehru Krishi Vishwavidyalaya (JNKVV, Jabalpur M.P.). This included important soybean cultivation areas covering 12 districts (viz, Jabalpur, Jabalpur sites, Jabalpur Khamariya, Ganjbasoda, Narshingpur, Narshingpur agriculture, Sagar, Gadarwara, Indore, Garhakota, Rewa and Tikamgarh region of Madhya-Pradesh, India as shown in Figure 1. 

Figure 1: Location of Study Sites (www.Mapsofindia.com).

Collection of Infected Soybean Crops

The present study carried out a comprehensive field survey to identify plants exhibiting characteristic symptoms of infection, such as wilting, stem discoloration, and necrosis. All the selected plants were representing a range of disease severity from different locations within the field. To capture variation in symptoms and pathogen distribution a detailed study was conducted. Disinfested tools were used such as pruning shears or scissors to minimize cross-contamination between plants (Bockus & Harvey, 2010). Sterilized tools were used between sampling locations or plants by dipping them in a disinfectant solution (e.g., 70% ethanol) to prevent spread of pathogens. In this point of view collecting symptomatic plant parts (stems, roots, leaves) that show clear signs of infection. All the samples included both diseased and apparently healthy plants for comparison and to assess pathogen distribution within the field. Sufficient samples were collected to ensure statistical reliability and representativeness (Frederiksen & Odvody, 1990). During the study period, samples were collected from a minimum of 10 to 20 plants per sampling site or field, depending on the size of the area and the uniformity of disease distribution. The plant samples were in clean, labeled bags or containers to prevent physical damage during transport. Keep samples cool and avoid extended exposure to sunlight or heat, especially during transit to the laboratory. Once the samples were reach the laboratory, process them promptly to prevent deterioration or loss of diagnostic value. Depending on the purpose (e.g., pathogen isolation and DNA extraction) were store samples appropriately (refrigeration, freezing or drying) until further analysis (Islam et al., 2017).

Isolation of pathogen and observation of their morphological characters

The pathogen Macrophomina phaseolina was isolated from infected plant tissues exhibiting characteristic symptoms such as charcoal rot, stem discoloration, and wilting. Infected root or stem segments were surface-sterilized using 1% sodium hypochlorite for 2 minutes, rinsed thrice with sterile distilled water, and then placed on Potato Dextrose Agar (PDA) medium under aseptic conditions. The inoculated plates were incubated at 28?±?2?°C for 5-7 days. Fungal growth was initially observed as fluffy white mycelium that gradually turned grey to black. The morphological identification was based on macroscopic and microscopic characters. Colonies of M. phaseolina appeared dark grey to black with abundant microsclerotia. Under the microscope, microsclerotia were spherical to oblong, dark brown to black, measuring 80-120?µm in diameter. Mycelium was septate, hyaline in the early stages, and turned pigmented with age. The identification was confirmed by comparing morphological traits with standard descriptions (Dhingra and Sinclair, 1978; Babu et al., 2011). Pure cultures were sub-cultured and stored for further pathogenicity and molecular characterization.

Slide Culture Technique

The slide culture technique is an essential method used in mycology to observe and document the microscopic morphological characteristics of fungal structures such as hyphae, conidia, and microsclerotia in their natural, undisturbed arrangement. This technique is especially valuable for delicate fungi like Macrophomina phaseolina, where microscopic features are critical for identification according (Dhingra et al., 2022).

Optimization of Various Conditions from Fungal isolates

The fungus is known for its wide host range and adaptability across diverse agro-climatic zones of Madhya Pradesh. The present study evaluates the influence of temperature, pH, and radial growth patterns of M. phaseolina isolates under controlled laboratory conditions. 

Effect of Temperature

It is well established phenomenon that the temperature significantly affects the biochemical activity of pathogens. 20 ml of PDA was poured in each of sterilized petri plates. Each petri plate was inoculated aseptically by placing a 5 mm disc in the center from actively growing 7 days old culture on PDA in petri dishes. The inoculated petri dishes were incubated at 10, 15, 20, 25, 30 and 35±1°C temperature for 96 hrs according to Srinivas et al., (2017).   

Effects of pH

The study was conducted to determine the impact of various pH levels on the growth of the fungus by measuring the effect of different hydrogen ion concentrations in the medium. The initial pH of the basal medium before autoclaving was adjusted 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0 and 8.5 with a maintained by using 0.1N NaOH or 0.1N HCl. After autoclaving the pH was again tested. The inoculated petri plates were incubated at 30±1°C for 96 hrs and observed the radial growth (mm) was recorded (c).  

Test the Efficacy of Fungicides against Macrophomina phaseolina in-vitro condition 

Various fungicides as listed below at different concentrations were evaluated for their effectiveness against pathogen by poisoned food technique as suggested by Nene and Thapliyal (1993). The fungicidal agents such as Azoxystrobin 23%, Carbendazim 50% and Propiconazole 25% were observed in this work. Stock solutions of each fungicide were prepared and aseptically incorporated into the sterilized PDA to achieve final concentrations of 25 ppm, 50 ppm and 100 ppm. The fungicide-amended media were then dispensed uniformly into sterile 90 mm Petri dishes within a laminar airflow cabinet to maintain aseptic conditions.  All plates were incubated in an incubator set at 28 ± 1°C for a duration of 7 days. Plates were positioned in an inverted manner to prevent condensation related contamination and growth interference.

Percent reduction (%) = (Dc-Tc) × 100

                                             Dc

Where:

Dc = Average diameter of the fungal growth in control.

Dt = Average diameter of the fungal growth treatment.

Table 1. Treatments detail to test the efficacy of fungicides against M. phaseolina in-vitro condition.

RESULT AND DISCUSSION  

In the present study investigations were undertaken to considering the economic significance of soybean charcoal rot disease, determine status, role of factors in disease development and management through integrating approaches. The results so obtained were presented here with.  

Survey and Collection of Infected Soybean host by the pathogens

A survey was conducted in soybean growing various districts of Madhya Pradesh during Kharif season 2016-17 and 2017-18 revealed that the charcoal rot caused by Macrophomina phaseolina (Tassi) Goid from agriculture fields. The soybean showing typical symptoms of charcoal rot at flowering and pod formation stage were collected from some districts of Madhya Pradesh state during second week of September to first week of October. All samples were brought in the laboratory condition and symptoms were recorded in detail Table 2. The roots of affected plant showing the symptoms of charcoal rot were cut and isolation were made for the presence or absence of the causal agent. Similarly, Cui et al., 2021 M. phaseolina isolates from soybean plants exhibiting symptoms of charcoal rot disease from different regions. Conventional identification methods, such as morphological and cultural characteristics, were employed to tentatively identify the isolates as M. phaseolina (Cui et al., 2021). Further confirmation was done through molecular techniques, including DNA sequencing and phylogenetic analysis (Cui et al., 2021).

Table 2. Survey, Collection, and Meteorological Evaluation of Soybean Fields Affected by Charcoal Rot in Madhya Pradesh (2017-2018).

The observed data from Table 3 and Figure 2 indicates that the mean disease incidence of soybean root rot across surveyed districts of Madhya Pradesh was 10.31% over the two seasons (Kharif 2016-17 and 2017-18). The disease incidence varied significantly between districts, highlighting geographic differences in disease pressure, which could be attributed to environmental factors, varietal susceptibility, and agronomic practices. The maximum mean disease incidence was recorded in Ganjbasoda (14.87%), followed by Gadarwara (14.55%) and Sagar (11.87%), all of which reported values above the district-level average of Narshingpur (10.88%).  In contrast, districts such as Indore (10.67%), Jabalpur (9.88%), Tikamgarh (9.72%), and Rewa (7.62%) reported disease incidences below the district mean. Jabalpur district appeared multiple times, with three observations showing slightly varying disease levels: 9.16%, 9.82%, and 9.88%. The lowest disease incidence was observed in Narshingpur II (7.18%), followed closely by Garhakota (7.57%) and Rewa (7.62%). These trends indicate that Ganjbasoda and Gadarwara are critical areas for root rot management interventions. The variations may be due to differences in microclimate, cropping systems, or pathogen load. This trend is consistent with previous research by Gupta et al. (1983), who documented root rot incidence in Northern Madhya Pradesh ranging from 3.58% to 20.63%, which is comparable to the present findings. 

Table 3. The disease incidence (%) of soybean in various districts of Madhya Pradesh.

Figure 2: Diseases Incidence of Soyabean in different regions of Madhya Pradesh.

Isolation of Pathogen and Observation the Morphological Characters

Isolation of pathogen morphological variability of M. phaseolina isolates 

The pathogen was successfully isolated from infected soybean samples using selective PDA media or appropriate isolation techniques based on the suspected type of pathogen (M. phaseolina). The pure cultures of the isolated pathogen were obtained through single-colony isolation methods. Macrophomina phaseolina (Tassi) Goid was isolated from the samples collected in surveyed chickpea growing blocks of Indore district of Madhya Pradesh were studied for their discernible characters such as the Colour of colony, colony diameter, Colour and shape of sclerotia, length and width of sclerotia and on the basis of above characters, 12 isolates were designated as (I_1, I_2, I_3……to I₁₂) localities cultural type. The isolated pathogen was cultured on specific growth media under controlled laboratory conditions (temperature, humidity and light or dark condition). Growth characteristics such as colony morphology (size, shape, color, texture) and growth rate with the different incubation time were observed and documented. Variations in cultural and morphological characteristics were recorded in all the isolates of Macrophomina phaseolina was result obtained as shown in Figure 3.

Figure 3. A depicted Image of Isolation and Identification of Macrophomina phaseolina.

Mycelia Development

During in this study, the results revealed that variation of morphological culture in mycelia growth of the Macrophomina phaseolina were isolated as given in Table 3 and Figure 2. The colony colour is greyish white in I₁, I₄ and I₅, grey with black margin in I₂, greyish white with grey margin in I₃, grey I₆ and greyish black colour in I₇. Hyphal morphology (e.g., septate, aseptate). The spore morphology (shape, size, color, presence of spore-bearing structures like conidia or sporangia).

Table 4: The Incubation Periods of soybean in various districts of Madhya Pradesh.

Figure 4: To study mean radial growth (mm) among the all isolates of Macrophomina phaseolina at 25 °C for different incubation periods.

The colony diameter and growth of different isolates of M. phaseolina were recorded at 48 hrs after incubation up to 120 hrs. Maximum radial growth were observed in isolates I₁, I₄, I₇ (79 mm) were completed their radial growth within 120 hrs of inoculation. Isolates I₇, I₈, I₁₁ recorded 79.2 mm, 84.4 mm, and 84.6 mm after 120 hrs of inoculation. Minimum radial growth was observed in isolates I₆. Growth even after 48, 72 and 120 hrs of inoculation period. Similarly, Waseer et al., (1990) reported that M. phaseolina from soybean grew best on potato dextrose agar or potato dextrose broth at 35°C. However, least growth occurred on either media at 20°C or 40°C. Suriachandraselvan and Seetharaman (2003) evaluated 5 culture media (potato dextrose agar, oat meal agar, Richard’s agar, Czapek’s dox agar and peptone sucrose agar) and they observed that potato dextrose agar was found best culture medium for mycelial growth and sclerotial formation of M. phaseolina. Sharma et al., (2004) reported potato dextrose agar medium as the best for mycelial growth and sclerotial formation of Rhizoctonia bataticola. Salunkhe et al., (2009) evaluated six media for growth of Rhizoctonia bataticola and stated that potato dextrose agar medium was the most suitable medium for growth and sclerotial formation of R. bataticola. Hooda and Grover (1982) studied the isolates of R. batalicola obtained from different plant species and plant parts of the same host which differed in their morphological and cultural characteristics. Gupta and Kolte (1982) also observed variation in growth characters, size and production of sclerotia, pycnidia and pycnidiospores in M. phaseolina.

Sclerotial growth

Table 5: Variation in abundance, growth, colour, shape and size of sclerotia of M. phaseolina isolates.

Figure 5: Variation in abundance, colour, shape and size of sclerotia of M. phaseolina isolates.

The colony diameter and growth of different isolates of M. phaseolina were recorded at 48 hrs after incubation up to 120 hrs. Maximum radial growth were observed in isolates I₁, I₄, I₇ (79 mm) were completed their radial growth within 120 hrs of inoculation. Isolates I₇, I₈, I₁₁ recorded 79.2 mm, 84.4 mm, and 84.6 mm after 120 hrs of inoculation. Minimum radial growth was observed in isolates I₆. Growth even after 48, 72 and 120 hrs of inoculation period. 

Effect of various temperature on growth of Macrophomina phaseolina

Temperature plays an important role in infection and disease development. Effect of different temperature viz., 10°C, 15°C, 20°C, 25°C, 30°C and 35°C on radial growth of M. phaseolina were studied in in-vitro condition and observation have been presented in Table 4 and illustrated in Figure 3. The maximum radial growth of the pathogen was recorded at 30°C (76.00 mm) which was significantly superior followed by 25°C (76.04 mm), 35°C (74.52 mm), 20°C (65.27 mm), 15°C (37.25 mm). Lowest radial growth was obtained at 10°C (25.45 mm) 120 hrs of incubation period. The maximum mycelial growth was observed at 35ºC followed by 30 and 25ºC in all the isolates. The optimum temperature for dry root rot severity rating was at 35°C (8.5) followed by 30°C (7.9) followed by 25°C (7.0).  Moreover, Parmar et al., (2018) tested different temperatures for M. phaseolina growth and found that temperature range of 25 to 35°C found optimum for growth and sclerotial formation. Whereas 30°C (79.44 mm) was ideal for growth of fungus. Sanjay et al., (2020a) reported that 30°C temperature was optimum for growth of Macrophomina phaseolina which was reduced growth significantly at below 25°C and above 35°C.   

Table 6: Effect of various temperatures on growth of Macrophomina phaseolina.

Figure 6: Effect of various temperatures on growth of Macrophomina phaseolina.